ABSTRACT In many bacteria, RNA-mediated regulation has been recognized to have a significant role in controlling virulence factors, combating stress responses, and adapting to environmental niches. Numerous examples of these mechanisms have been characterized, including small base-pairing RNAs (sRNAs), riboswitches, and RNA-based thermosensors. Borrelia burgdorferi, the causative agent of Lyme disease, is a particularly unique bacterial pathogen. It has a complex lifecycle in which the bacterium absolutely relies on a tick vector and mammalian reservoirs, for transmission and nutrient acquisition. Given that B. burgdorferi successfully evades diverse host immune responses and can survive in differential growth conditions, the bacterium must harbor various mechanisms to control genes required for metabolism and infectivity. Despite this, few regulators have been identified in B. burgdorferi to explain changes in gene expression. Converging evidence suggests that RNA-mediated post-transcriptional mechanisms impact B. burgdorferi environmental adaptation. This includes documentation of novel un-annotated transcripts from across the transcriptome, such as numerous sRNAs and long 5? untranslated mRNA regions. A homolog of the RNA chaperone Hfq, which in many bacteria is considered the ?master regulator? of post-transcriptional regulation, has also been identified in B. burgdorferi along with one Hfq-dependent sRNA. This proposal will utilize global and targeted approaches to characterize new mechanisms of RNA-mediated gene regulation in the Lyme disease pathogen. RNA-seq methods will be carried out to create a new annotation of the transcriptome, identify RNAs which control transcription of biologically relevant genes, and map direct base-paring RNAs to their specific targets. Specific sRNAs will be extensively characterized and a variety of RNA biochemistry experiments will be performed to define the mechanisms for how these putative regulators affect their mRNA targets. With a growing incidence of Lyme disease, it is critical to identify and characterize new principles of B. burgdorferi gene regulation to ultimately achieve a more comprehensive understanding of basic biology, host adaptation, and infectivity.